Vol.

166.

January

No. 30,

BIOCHEMICAL

2. 1990

AND

BlOPHYSlCAL

RESEARCH

COMMUNICATIONS

Pages

1990

595-600

TEE INFfIBITION OF ED?lANNElJTROPBILELASTASEAND CATREPSINC BY PEPTIDYL 1,ZDICARBONYL DERIVATIVES Shujaath

Mehdi’,

Merrell

Michael R. Angelastro, Joseph P. Burkhart, Norton P. Peet, and Philippe Bey

Dow Research

Received

November

Institute,

2110 E. Galbraith

Road,

Jack

R. Koehl,

Cincinnati,

OH 45215

9, 1989

Summary: Neutrophil elastase and cathepsin G are serine proteases that can damage connective tissue and trigger other pathological reactions. Compounds specificity and bearing an LXcontaining a peptide sequence to impart dicarbonyl unit (a-diketone or a-keto ester) at the carboxy terminus are potent inhibitors of the neutrophil serine proteases (human neutrophil elastase: R-Val-COCH,, Ki = 0.017 uM; R-Val-COOCH,, K, = 0.002 uM; human neutrophil cathepsin G: R-Phe-COCH,, Ki = 0.8 uM; R-Phe-COOCH,, Ki = 0.44 uM; R = N-(4-[(4-chlorophenyl)sulfonylaminocarbonyl]phenylcarbonyl)ValylProlyl).

Neutrophils immune

are

to arrive

system

response

hydrolytic

species.

The

interface

between

extracellular the

serine

implicated. 7),

dicarbonyl

signal

the cell

beneficial for derivatives

and cathepsin

large,

in

I and

are

a

report

excellent

cells

G.

are

or

of the trauma

oxygen

phagolysosome,

at

or into

the neutrophil

under

here inhibitors

the the

granules

Since these enzymes of the two enzymes where

in

of neutrophils reactive

substrate, of

conditions

inhibitors We

II

into

inhibition

pathological

of elastase

granules generate

proteases

and cathepsin

emphysema.

can

noningestible

proteins,

first

inflammation

released

neutral

elastase

and other

that

the

among

The lysosomal

are a

are

infection,

enzymes

The major

proteases

of

(1,2).

and and

that

site

contents

Many types

particularly

elastase

granule

tissue

therapeutically

a

enzymes

milieu.

connective

leukocytes

at

to a chemotactic

contain

are

short-lived

degrade may

neutrophils

--in vivo evaluation that the new peptidyl

be

are (3a-

of human neutrophil

G.

MATERIALSAND HEXHODS The a-diketone and a-keto ester derivatives of Nprotected ValProVal(compounds Ia and Ib, Figure 1) and ValProPhe(IIa and IIb, Figure 1) were synthesized by the application of methods reported elsewhere (8-11). Compounds Ia, IIa and IIb are approximately equal mixtures of diastereomers (epimeric at the a-carbon center next to the a-dicarbonyl unit); Ib is predominantly a single diastereomer with the L configuration at the valyl a-carbon. Common biochemicals and the chromogenic substrates N*To whom correspondence

should

be addressed. 0006.291x/90 59.5

$1.50

Copyright 0 1990 by Academic Press. Inc. All rights of reproduction in any form reserved.

Vol.

166,

No.

2, 1990

BIOCHEMICAL

R-NH

1.

BIOPHYSICAL

R’

RESEARCH

COMMUNICATIONS

R-NH

0

Figure

AND

10

R’=

0 ,&,,

Ib

R’=

AOCH

Structures

3

3

0

IIo

R’=

0 ACH

IIb

R’=

A,,,

of compounds

used

3

3

in this

study.

and N-Sue-AlaAlaProPhe-e-nitroanilide were MeOSuc-AlaAlaProVal-e-nitroanilide Azure hide powder was from Calbiochem. from Sigma. elastase and cathepsin G were purified Enzyme Isolation Human neutrophil Frozen purulent sputum (20 g) was quickly from human purulent sputum (12). thawed in 25 mL of 0.34 M sucrose and the mixture was homogenized at high speed in a glass/teflon homogenizer. DNAse (Sigma; 10,000 units) was added and the homogenate was stirred for 30 min at 4OC. The mixture was centrifuged at 1OOOg for 10 min, the pellet was homogenized again and the homogenate was centrifuged at 1OOOg for 10 min. The supernatants from each step were pooled and The 30,OOOg pellet was washed three times centrifuged at 30,OOOg for 30 min. by resuspension in 0.34 M sucrose and centrifugation. The washed pellet was suspended in 0.05 M sodium acetate (NaOAc), pH 5.5, containing 1 M sodium chloride (NaCl) and 0.1% Brij 35. The suspension was sonicated in an ultrasonic The suspension was centrifuged at 30,OOOg and cleaning bath for 30 min at 4°C. the supernatant (granule extract) was saved. To partially purify elastase, the granule extract was diluted with 9 ~01s. of 0.05 M NaOAc, pH 5.5, containing 0.1 M NaCl and the cloudy solution was applied to a column (2.6 cm x 26 cm) of carboxymethyl-Trisacryl (LKB). The column was first washed with the diluting buffer, and then elastase was eluted with 0.05 M NaOAc (pH 5.5)/0.45 M NaCl directly in For the purification of cathepsin G , sputum was homogenized (13). The supernatant after 0.05 M NaOAc (pH 5.5)/1.0 M NaCl/O.l% Brij 35. centrifugation at 30,OOOg for 30 min was diluted 15-fold with cold deionized The precipitated protein was water and the mixture was kept at O°C for 30 min. recovered by centrifugation and redissolved in 0.05 M NaOAc (pH 5.5)/0.6 M 30 min, cathepsin G was isolated NaCl. After centrifugation at 30,OOOg for from the supernatant by cation exchange chromatography using the Pharmacia FPLC sys tern. A portion (10 mL) of the supernatant was applied to a Mono S column (1 cm x 10 cm; Pharmacia) which was washed at 2 mL/min with 60 mL of 0.6 M NaCl in NaOAc buffer and then eluted with a linear gradient over 56 min of 0.6 M to 1.0 M NaCl in 0.05 M NaOAc (pH 5.5). Isozymes of cathepsin G eluted between isozyme peak (at 0.80 M NaCl) was used for 0.78 M and 0.86 M NaCl. The first the experiments described here. The protein was a single band by SDS-PAGE, and further confirmed by amino-terminal amino acid its purity and identity were analysis (14). the specific substrate N-MeOSucE;lzyme Assays. Elastase was assayed using The assays were AlaA aProVa -p-nltroanllrde (15,16) (K, = 0.16 f .04 mM). carried out at 37’C in 3.0 mL of 0.1 M HEPES (pH 7.5), 0.5 M NaCl and 0.1% Brij Substrate and inhibitor were added from stock solutions in DMSO, and the 35. total DMSO in the reaction mixture was 10% v/v. The reaction was monitored at 410 nm using an HP8452 (Hewlett Packard) spectrophotometer (c,,, nl = 9.16 mH-l conditions). Approximately 2 pg of cm-l for e-nitroaniline under the assay protein of specific activity 3.6 units/mg was used in each assay (1 unit is the of 1 umole per minute of N-MeOSucamount of protein catalyzing the hydrolysis AlaAlaProVal-P-nitroanilide at saturating concentration under the assay conditions described above). 596

Vol.

BIOCHEMICAL

166, No. 2, 1990

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Cathepsin G was assayed using N-Sue-AlaAlaProPhe-p-nitroanilide as the substrate (15,17) under the conditions described above for elastase (K, = 5.6 f 1 mM). Approximately 0.1 ug of protein of specific activity 40 units/mg was used in each assay (1 unit is as defined above for elastase except that the substrate is different). Kinetic constants were determined from double reciprocal plots using the kinetics software of the HP8452 spectrophotometer. The hydrolysis of azure hide powder was carried out by incubating 15 mg of hide powder in 1.0 mL of the buffer used in the spectrophotometric assays, 10 uL of granule extract (containing 0.23 units of elastase and 0.13 units of cathepsin G), and inhibitor added as a DMSO solution (final DMSO concentration = 3% v/v). The mixture was incubated at 37OC in a shaking water bath and the contents were mixed every 15 min. After 1 hr, the reaction mixture was filtered through glass wool and the absorbance at 595 nm of the filtrate was measured. Bovine a-chymotrypsin (Sigma) was assayed using N-Suc-AlaAlaProPhe-pnitroanilide as the substrate and bovine thrombin (Sigma) was assayed using DPhePipecolylArg-P-nitroanilide (Kabi, S-2238) as the substrate (18). The use of TRIS buffer was avoided and instead HEPES buffer was used. A fluorometric assay was used to measure enkephalinase (endopeptidase 24.11) activity using Dansyl-D-AlaGly(p-nitro)PheGly-OH (Sigma) as the substrate (19). The enzyme was purified from rat kidneys by extraction of the microvilli fraction with Triton X-100 (20) followed by anion exchange chromatography using a Mono 0 column (Pharmacia). Calpain was isolated from chicken gizzard smooth muscle and assayed using a fluorogenic substrate as described before (21). RESDLTS

peptides

AND

are

cysteine

protease

made,

proteases

the

led

since

proteases

(10,27).

in protease showed

Other

that

work a-keto

was first many

esters

are

~~m;,~.

bzu,ble

reciprocal

groups ester by

plot

Powers

inhibitors

10

of

group

of

serine

and

to

the

a hemiacetal

principle

containing poor

including

good

form

this

Our interest

are

a-keto

1 /[S],

to

utilize

described

0

nucleophile

(7,25,26).

inhibitors, also

inhibit

site

inhibitor

ketones

derivatives

aldehydes

active

that

functional

fluoro

The use of the to

the

ketones other

peptidyl

inhibitors this

of

the

inhibitors

fluoromethyl

aldehyde

Peptidyl

of

carbonyl

us to explore

carbonyl,

(22).

addition

(23,24).

notably

extended

by

aldehyde

hemithioacetal

naturally-occurring

inhibitors

proteases

electrophilic

Some

DISCUSSION

or

have in

been

cysteine

an electrophilic

inhibitors

of cysteine

as an electrophilic

unit

and co-workers those

described

of cysteine

(28);

we

here,

proteases

and (10).

20

mM-’ initial

597

rate

data

for

the

inhibition

of

Vol.

166,

No.

We also

2, 1990

described

serine

the

protease

work

to inhibitors of elastase

in

the

and

the

is

Our is

cysteine G.

of

the on

[by

sequence

within

comparison a series

we adopted

the

of

calpain work

that

(31,32).

Inhibition

constants

neutrophil

elastase

compounds

for

and

within

competitive

inhibitors

binding

behavior:

assays

(30 min),

competitive

competitive

a series substrate

elastase,

the extent

inhibition

addition,

Ia is

proteases),

(Table

smaller

much

potent

not

extent

inhibitor

a

than

it

380,000

M-is-l

better for

of

is

poor

a

poor

the

of

the optimal protecting by Trainor

esters shown

for

compounds over

initial

human

in Table

and IIa

1. The

and IIb

exhibited the

data

are slow-

period

rate

inhibitor of

of

the

indicated

a-chymotrypsin

G inhibitor

IIa

cathepsin (Table

of cathepsin

of elastase and

with

substrate for chymotrypsin

inhibitor

protease),

inhibits

of a-chymotrypsin

N-Sue-AlaAlaProPhe-e-nitroanilide considerably

the

was constant

inhibitor

cysteine

The cathepsin

2).

G are

plots Ia

is

good (a

of p-

2).

IIa

a

calpain

protease)

inhibition

a-diketone

G inhibitor

a-keto

of elastase None of

reciprocal

(Figure

The elastase-specific cathepsin

of

and double

cathepsin

G.

the

(30)

the N-terminal

and

inhibitors

of cathepsin

for

used

a-diketones

and human neutrophil

Ia and Ib are

inhibitors

to be N-MeOSuc-AlaAlaProVal-,

-ValProVal-

the

The

approach

-ValProPhe-

4-[(4-chlorophenyl)sulfonylaminocarbonyl]phenylcarbonyl

and co-workers

(10).

was in progress,

best

For

(15)

of the

a-diketone

the

(25)].

by Powers

sequence

inhibitors

a-diketone

sequence was

was found

shorter

this

of peptidyl

finding

k,,,/K,

in

of the

peptide the

COMMUNICATIONS

protease While

and uses

based

group

the application

synthesis use

RESEARCH

a-diketone

and cathepsin

(29).

BIOPHYSICAL

N-Sue-ValProPhe-e-nitroanilide G

the

AND

of

report

the

G inhibitors

cathepsin

group

this

reported

nitroanilides,

but

use

of elastase

et al.

cathepsin

novel

a-chymotrypsin

we describe

Stein

BIOCHEMICAL

G

2). a

chymotrypsin and 1,100

and

but

partially than

thrombin

for

In

(serine

(a

this

metal10 to a

compound

(25),

the

similar for

1).

enkephalinase

Analogously

M-is-l

(Table

enkephalinase inhibits

G, and

sequence

cathepsin

G (k,,,/K,

human

is

a

substrate

cathepsin

is

a = G)

(15,30). The cathepsin

inhibition

constants

G were

TABLE 1.

also

for

the

measured

Inhibition neutrophil

constants elastase

two inhibitors Ia and IIa the granule extract using

(Ki, MM) for and cathepsin Ia

Ib

the inhibition G by I and II IIa

for elastase containing

of human

IIb

Enzyme Human elastase Human cathepsin

G

0.017 >lOOO

598

0.002

-

>350 0.8

0144

and both

Vol.

166,

No.

2, 1990

BIOCHEMICAL

TABLE 2.

Inhibition

AND

BIOPHYSICAL

of several (Ki values,

RESEARCH

proteases uM)

COMMUNICATIONS

by Ia and IIa

IIa

Ia Enzyme >200 >lOO >lOOO >600

Bovine a-chymotrypsin Rat enkephalinase Chicken calpain Bovine thrombin

enzymes

and

(elastase, with

Ia:

are

examined

hydrolysis

cathepsin

by JO%.

together,

the

at 50 uM

saturated

at

not

inhibitors cathepsin directly

At

a

decrease are

lower the

potent

any

by

or indirectly

of in

by harmful

a

azure

releases

soluble

2

50 50

8 6 II $ 9 2

the

number

neutrophils

uM

or

uM of each

>90%.

Each that

IIa 50/LM

and thus

a

The

100 uM reduced

the

inhibitor

present is

enzyme

inhibitor;

a higher

the

peptidyl

a-dicarbonyl

neutrophil

serine

of

situations

equally

is already

concentration

and

mediate

DLIIaIa IO lOO!.dl

599

50/A

100/d

derivatives

proteases.

in which effects

f

NO Inhibitor

powder.

3).

the

0.4

00 /

the

50 pM or 100 uM,

inhibitor

each

to the host.

0.2

hide

by 20% (Figure

0.8

0.6

and that

prevent

peptides,

of either

1.0 E c

the p-

further. that

inhibitors

released

substrate,

suggesting

demonstrated

be useful

two enzymes,

weight

of

of

hydrolysis

the

closely

that

to

of

reduced uM,

suggesting

inhibitors

concentration

100

for

concentration

concentration

at

measured

Ki = 1 uM) agree

the hydrolysis

concentration

we have

should G are

molecular

reduced

was

or

IIa: enzymes,

synthetic

At a

a

hydrolysis

In summary, I and II

at

G,

values

Ki

extract.

the

preparation IIa

Ia

effective did

of

The

specific

the crude high

G inhibitor

the

indeed

in solution.

inhibitor

hydrolysis

in

collagen

appears

cathepsin the purified

are

ability

insoluble

of this

color

elastase

stable

the

substrates.

using

used

the

of

Proteolysis blue

measured

substrates

inhibitors We

the

p-nitroanilide Ki = 0.017 uM and

the Ki values

nitroanilide the

the

using

0.009 -40 >50 >600

Iotira 50@4 each

that

These

elastase are

and either

Vol.

166, No. 2, 1990

BIOCHEMICAL

AND BIOPHYSICAL

ACKNOWLEDGlfENFS We are grateful to Dr. Frank human purulent sputum and to Dr. Eugene Giroux for

RESEARCH COMMUNICATIONS

Kellog for providing many discussions.

us with

REFERENCRS 1. Lab. Investigation 59, 300-320. Sandborg, R. R., and Smolen, J. E. (1988) 2. Travis, J. (1988) Am. J. Med. 84(Suppl 6A), 37-42. 3. Bonney, R. J., Ashe, B., Maycock, A., Dellea, P., Hand, K., Osinga,D., Fletcher, D., Mumford, R. Davies, P., Frankenfield, D., Nolan, T., Schaeffer, L., Hagmann, W., Finke, P., Shah, S., Dorn, C., and Doherty, J. (1989) J. Cell Biochem. 39, 47-53. 4. Hassall, C. H., Johnson, W. H., Kennedy, A. J., and Roberts, N. A. (1985) FEBS Lett. 182, 201-295. 5. Nick, H. P., Probst, A., and Schnebli, H. P. (1988) Adv. Exp. Med. and Biol. 240, 83-88.A 6. Fournel, M. A., Newgren, J. O., Betancourt, C. M., and Irwin, R. G. (1988) Am. J. Med. 84(Suppl 6A), 43-47. 7. Trainor, D. A. (1987) Trends. Pharm. Sci. 8, 303-307. 8. Burkhart, J. P., Peet, N. P., and Bey, P. (1988) Tetrehedron Lett. 29, 3433-3436. 9. Angelastro, M. R., Peet, N. P., and Bey, P. (1989) J. Org. Chem. 54, 39133916. 10. Angelastro, M. R., Mehdi, S., Burkhart, J. P., Peet, N. P., and Bey, P. (1989) J. Med. Chem., in press. 11. Angelastro, M. R., manuscript in preparation. 12. Twumasi, D. Y., and Liener, I. E. (1977) J. Biol. Chem. 252, 1917-1926. 13. Baugh, R. J., and Travis, J. (1976) Biochemistry 15, 836-841. 14. Salvesen, G., Farley, D., Shuman, J., Przybyla, A., Reilly, C., and Travis, J. (1987) Biochemistry 26, 2289-2293. 15. Nakajima, K., Powers, J. C., Ashe, B. M., and Zimmerman, M. (1979) J. Biol. Chem. 254, 4027-4032. 16. Barrett, A. J. (1981) Methods in Enzymology 80, 581-588. 17. Barrett, A. J. (1981) Methods in Enzymology 80, 561-565. 18. Methods of Enzymatic Analysis (1984), Bergmeyer, H. U. Ed. in Chief, Verlag Chemie, Weinheim. 19. Florentin, D., Sassi, A., and Roques, B. P. (1984) Anal. Biochem. 141, 6269. 20. Malfroy, B., and Schwartz, J-C. (1984) J. Biol. Chem. 259, 14365-14370. 21. Mehdi, S., Angelastro, M. R., Wiseman, J. S., and Bey, P. (1988) Biochem. Biophys. Res. Commun. 157, 1117-1123. 22. Aoyagi , T., and Umezawa, H. in Proteases and Biological Control (1975), Reich, E., Rifkin, D. B., and Shaw, E. Eds., Cold Spring Harbor Laboratory. 23. Bendall, M. R., Cartwright, I. L., Clark, P. I., Lowe, G., and Nurse, D. (1971) Eur. J. Biochem. 79, 201-209. 24. Delbaere, L. T. J., and Brayer, G. D. (1985) J. Mol. Biol. 183, 89-103. 25. Imperiali, B., and Abeles, R. H. (1986) Biochemistry 25, 3760-3767. 26. Peet, N. P., Burkhart, J. P., Angelastro, M. R., Giroux, E. L., Mehdi, S., Kolb, M., Neises, B., Schirlin, D., and Bey, P. J. Med. Chem., in press. 27. Smith, R. A., Copp, L. J., Donnelly, S. L., Spencer, R. W., and Krantz, A. (1988) Biochemistry 27, 6568-6573. 28. Hori, H., Yasutake, A., Minematsu, Y., and Powers, J. C. (1985) Proceedings of the 9th American Peptide Symposium, 819-822. 29. Stein, M. M., Wildonger, R. A., Trainor, D. A., Edwards, P. D., Yee, Y. K ., Lewis, J. J., Zottola, M. A., Williams, J. C., and Strimpler, A. M. (1989) Poster presented at the 11th American Peptide Symposium, La Jolla, CA. 30. Tanaka, T., Minematsu, Y., Reilly, C. F., Travis, J., and Powers, J. C. (1985) Biochemistry 24, 2940-2047. 31. Krell, R. D., Stein, R. L., Strimpler, A. M., Trainor, D., Edwards, P., Wolanin, D., Wildonger, R., Schwartz, J., Hesp, B., Giles, R. E., and Williams, J. C. (1988) FASEB J. 2, A346. 32. Trainor, D. A., Bergeson, S. H., Schwartz, J. A., Stein, M. A., Wildonger, R. A., Edwards, P. D., Shaw, A., and Wolanin, D. J. (1986) European Patent Application 018930582. 600